Molecular Characterization of the Inositol 1,4,5-Trisphosphate Receptor Pore-forming Segment

Abstract

Specific residues in the putative pore helix, selectivity filter, and S6 transmembrane helix of the inositol 1,4,5-trisphosphate receptor were mutated in order to examine their effects on channel function. Mutation of 5 of 8 highly conserved residues in the pore helix/selectivity filter region inactivated the channel (C2533A, G2541A, G2545A, G2546A, and G2547A). Of the remaining three mutants, C2527A and R2543A were partially active and G2549A behaved like wild type receptor. Mutation of a putative glycine hinge residue in the S6 helix (G2586A) or a putative gating residue at the cytosolic end of S6 helix (F2592A) had minimal effects on function, although channel function was inactivated by G2586P and F2592D mutations. The mutagenesis data are interpreted in the context of a structural homology model of the inositol 1,4,5-trisphosphate receptor.Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) 3 are tetrameric ligand-gated ion channels located in the endoplasmic reticulum membrane that are co-activated by IP 3 and Ca 2ϩ (reviewed in Ref. 1). The Ca 2ϩ released through these channels can activate a diverse array of physiological processes, depending on the amplitude and frequency of Ca 2ϩ release (2). The IP 3 R contains a ligand-binding domain in the N-terminal region (amino acids 226 -576) and six transmembrane segments (S1-S6; amino acids 2276 -2590) in the C-terminal region of the receptor (3, 4). Several key aspects of IP 3 R function remain to be addressed, including the identification of critical residues that line the channel conduction pore and the exact mechanism of the IP 3 -activated gating process.In common with many voltage-gated ion channels, the IP 3 R and its close relative the ryanodine receptor (RyR) contain a short luminal pore helix and a selectivity filter (1). In the IP 3 R, these are located between the S5 and S6 helices. Experimental evidence suggests that the minimal channel domain of the IP 3 R lies in the portion of the receptor encoded by the sequences distal to the S5 helix (5). Previous mutagenesis studies of this region of the IP 3 R have focused on the selectivity filter sequence 2547 GVGD 2550 (numbering according to rat type-I IP 3 R). The results showed that the V2548I mutation increased conductance through the channel (6, 7), that the D2550A mutation inactivated channel function (6), and that the D2550E mutation decreased the divalent cation selectivity of the channel (6). Examination of the sequences of all three IP 3 Rs and RyR isoforms from several species in the region of the putative pore helix and selectivity filter indicates the presence of 8 highly conserved residues. In the present study, we have examined the effect of mutating these conserved amino acids on IP 3 R function.The N-terminal ligand-binding domain and the channel domain of the receptor show a direct intersubunit interaction in the tetrameric channel (8). A model for the gating mechanism of the IP 3 R channel has been proposed in which the N-terminal region of the receptor interacts with the channel domain ...

IP3 Receptors: Toward Understanding Their Activation

“…This model for the IP 3 R pore, where TMD5 (the outer helix) and TMD6 (inner helix) cradle a short pore helix and selectivity filter (Fig. 2C), is consistent with mutagenesis of residues within this region affecting ion permeation (Boehning et al 2001b;Dellis et al 2006;Dellis et al 2008;Schug et al 2008), with biophysical evidence that the narrowest region of the pore lies close to the luminal entrance of the RyR and with the intermediate resolution structures of the pore region of RyR1 (Samso et al 2009). A conserved acidic residue (D2550 in IP 3 R1) at the luminal end of the selectivity filter (Fig.…”

“…The changes in pore structure that allow it to open are also minimally understood. Indeed, mutation of the conserved Gly within TMD6 of IP 3 R (G2586 in IP 3 R1), which might have been thought to provide the gating hinge (Samso et al 2009), appears not to prevent IP 3 from opening IP 3 R (Schug et al 2008). In short, aside from knowing the regions of primary sequence that form the IP 3 R pore (TMD5-6) and a rather vague notion that its structure perhaps resembles that of K þ channels, we have only the most rudimentary knowledge of the structural determinants of how the IP 3 R pore opens and selects between ions.…”

Identification of Functionally Critical Residues in the Channel Domain of Inositol Trisphosphate Receptors

“…Transfection of Cells and Preparation of Microsomal Vesicles-COS-7 cells were grown in 150-mm plates and were cotransfected with DNA encoding IP 3 R (10 g) and SERCA2b (10 g) using LT-1 (Mirus) and Novafector (VennNova, Inc.) transfection reagents as described previously (29). Transfected cells were washed twice in PBS and scraped into isolation buffer (320 mM sucrose, 20 mM K-HEPES, pH 7.8, 0.5 mM EGTA, and 0.5 mM DTT).…”

Emergence of ion channel modal gating from independent subunit kinetics

“…Several mutations near the selectivity filter have been shown to inactivate the type 1 IP 3 R (55). If the methods of ref.…”

“…48, which constructed channels with mutations in the IP 3 binding domain of a known number of subunits, can be extended to the mutations identified by ref. 55, then the model makes strong, parameter-free predictions of channel behavior. With one defective subunit, the model predicts a channel that gates only in I and L modes, given that the H mode requires four subunits.…”